Critical contribution of OX40 ligand to T helper cell type 2 differentiation in experimental leishmaniasis

Infection of inbred mouse strains with Leishmania major is a well characterized model for analysis of T helper (Th)1 and Th2 cell development in vivo. In this study, to address the role of costimulatory molecules CD27, CD30, 4-1BB, and OX40, which belong to the tumor necrosis factor receptor superfamily, in the development of Th1 and Th2 cells in vivo, we administered monoclonal antibody (mAb) against their ligands, CD70, CD30 ligand (L), 4-1BBL, and OX40L, to mice infected with L. major. Whereas anti-CD70, anti-CD30L, and anti-4-1BBL mAb exhibited no effect in either susceptible BALB/c or resistant C57BL/6 mice, the administration of anti-OX40L mAb abrogated progressive disease in BALB/c mice. Flow cytometric analysis indicated that OX40 was expressed on CD4(+) T cells and OX40L was expressed on CD11c(+) dendritic cells in the popliteal lymph nodes of L. major-infected BALB/c mice. In vitro stimulation of these CD4(+) T cells showed that anti-OX40L mAb treatment resulted in substantially reduced production of Th2 cytokines. Moreover, this change in cytokine levels was associated with reduced levels of anti-L. major immunoglobulin (Ig)G1 and serum IgE. These results indicate that anti-OX40L mAb abrogated progressive leishmaniasis in BALB/c mice by suppressing the development of Th2 responses, substantiating a critical role of OX40-OX40L interaction in Th2 development in vivo.     

OX40 (CD134) controls memory T helper 2 cells that drive lung inflammation

Asthma is caused by memory Th2 cells that often arise early in life and persist after repeated encounters with allergen. Although much is known regarding how Th2 cells develop, there is little information about the molecules that regulate memory Th2 cells after they have formed. Here we show that the costimulatory molecule OX40 is expressed on memory CD4 cells. In already sensitized animals, blocking OX40-OX40L interactions at the time of inhalation of aerosolized antigen suppressed memory effector accumulation in lung draining lymph nodes and lung, and prevented eosinophilia, airway hyperreactivity, mucus secretion, and Th2 cyto-kine production. Demonstrating that OX40 signals directly regulate memory T cells, antigen-experienced OX40-deficient T cells were found to divide initially but could not survive and accumulate in large numbers after antigen rechallenge. Thus, OX40-OX40L interactions are pivotal to the efficiency of recall responses regulated by memory Th2 cells.     

Mature dendritic cells induce tumor-specific type 1 regulatory T cells

The aim of this study was to compare the tumor antigen-specific T-cell repertoire generated by transduced, human dendritic cells (DCs). The transductions were three commonly used antigen delivery procedures: adenovirus (AdV) infection, RNA electroporation, and liposome-mediated protein transfection. The DCs in each experimental group were transfected with similar efficacy and matured using TNF-alpha, anti-CD40, or lipopolysaccharide. Regardless of the gene transfer method or the maturation stimuli used, the DCs were indistinguishable with regard to surface phenotype and allostimulatory capacity. With the exception of the Adv transduced group, the T cells generated were tumor antigen specific, as characterized by high IFN-gamma production. The T cells generated upon stimulation with DCs subjected to AdV infection, and subsequently treated with TNF-alpha, exhibited tumor antigen specificity, but accompanied by reduced proliferation and IFNgamma production and increased IL-10 production. Moreover, these T cells exerted a suppressive effect on both autologous and allogeneic lymphocytes resembling type 1 regulatory T cells (Tr1). The authors show that mature DCs may induce tumor antigen-specific Tr1 cells by the appearance of high IL-10 and low IL-12. Similar results were also obtained with AdV-infected and TNF-matured DCs regardless of the transgene used. This work supports the conclusion that it can no longer be assumed that mature DCs induce only antitumor reactive T cells.     

Human monocyte-derived dendritic cells induce naive T cell differentiation into T helper cell type 2 (Th2) or Th1/Th2 effectors. Role of stimulator/responder ratio

The subset of dendritic cells (DCs) and the nature of the signal inducing DC maturation determine the capacity of DCs to generate polarized immune responses. In this study, we show that the ability of human monocyte-derived DCs (myeloid DC(1)) to promote T helper type 1 (Th1) or Th2 differentiation was also found to be critically dependent on stimulator/responder ratio. At a low ratio (1:300), mature DCs that have been differentiated after inflammatory (Staphylococcus aureus Cowan 1 or lipopolysaccharide) or T cell-dependent (CD40 ligand) stimulation induced naive T cells to become Th2 (interleukin [IL]-4(+), IL-5(+), interferon gamma) effectors. Th2 differentiation was dependent on B7-CD28 costimulation and enhanced by OX40-OX40 ligand interactions. However, high DC/T cell ratio (1:4) favored a mixed Th1/Th2 cell development. Thus, the fact that the same DC lineage stimulates polarized Th1 or Th2 responses may be relevant since it allows the antigen-presenting cells to initiate an appropriate response for the signal received at the peripheral sites. Controlling the number and the rate of DC migration to the T cell areas in lymphoid tissues may be important for the therapeutic use of DCs.     

Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization

Pollen grains induce allergies in susceptible individuals by release of allergens upon contact with mucosal membranes of the upper respiratory tract. We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids that attract cells involved in allergic inflammation such as neutrophils and eosinophils. Here we demonstrate that soluble factors from birch (Betula alba L.) pollen activate human dendritic cells (DCs) as documented by phenotypical and functional maturation and altered cytokine production. Betula alba L. aqueous pollen extracts (Bet.-APE) selectively inhibited interleukin (IL)-12 p70 production of lipopolysaccharide (LPS)- or CD40L-activated DC, whereas IL-6, IL-10, and TNFalpha remained unchanged. Presence of Bet.-APE during DC activation resulted in DC with increased T helper type 2 (Th2) cell and reduced Th1 cell polarizing capacity. Chemical analysis of Bet.-APE revealed the presence of phytoprostanes (dinor isoprostanes) with prostaglandin E(1)-, F(1)-, A(1)-, or B(1)-ring systems of which only E(1)-phytoprostanes dose dependently inhibited the LPS-induced IL-12 p70 release and augmented the Th2 cell polarizing capacity of DC. These results suggest that pollen-derived E(1)-phytoprostanes not only resemble endogenous prostaglandin E(2) structurally but also functionally in that they act as regulators that modulate human DC function in a fashion that favors Th2 cell polarization.     

Lentiviral vector-transduced dendritic cells induce specific T cell response in a nonhuman primate model

Dendritic cells (DCs) are effective in stimulating and controlling the outcome of T cell responses. Human immunodeficiency virus type 1-based lentiviral vectors can achieve sustained transduction of genes/antigens in dividing and nondividing cells, thus representing a candidate vector for stable expression of antigens in DCs. We previously established conditions for transduction of purified cytokine mobilized rhesus CD34(+) cells in vitro, and transplantation of the autologous transduced cells in a nonhuman primate model in vivo. In the present study, we transplanted DCs derived from EGFP-transduced CD34(+) cells into nonmyeloablated rhesus macaques. Transplantation of DCs stably expressing EGFP into autologous animals induces persistent, long-lived (up to 100 weeks) EGFP-specific T cell responses. Of note, no humoral responses against EGFP are detected in the transplanted animals. These studies provide, to our knowledge, the first demonstration that lentiviral transduction of CD34(+) progenitor cells subsequently differentiated to DCs is capable of priming a specific T cell response in a nonhuman primate in vivo. Taken together, our data provide formal in vivo evidence that lentivirus-transduced dendritic cells represent a potential approach in eliciting cellular immune responses in primates.     

Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells.

Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c(+) DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98-100% pure CD11c(+) DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide-specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-gamma (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-gamma production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.     

Inhibition of T helper cell type 2 cell differentiation and immunoglobulin E response by ligand-activated Valpha14 natural killer T cells.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.     

Human dendritic cells activated by TSLP and CD40L induce proallergic cytotoxic T cells

Human thymic stromal lymphopoietin (TSLP) is a novel epithelial cell-derived cytokine, which induces dendritic cell (DC)-mediated CD4+ T cell responses with a proallergic phenotype. Although the participation of CD8+ T cells in allergic inflammation is well documented, their functional properties as well as the pathways leading to their generation remain poorly understood. Here, we show that TSLP-activated CD11c+ DCs potently activate and expand naive CD8+ T cells, and induce their differentiation into interleukin (IL)-5 and IL-13-producing effectors exhibiting poor cytolytic activity. Additional CD40L triggering of TSLP-activated DCs induced CD8+ T cells with potent cytolytic activity, producing large amounts of interferon (IFN)-gamma, while retaining their capacity to produce IL-5 and IL-13. These data further support the role of TSLP as initial trigger of allergic T cell responses and suggest that CD40L-expressing cells may act in combination with TSLP to amplify and sustain pro-allergic responses and cause tissue damage by promoting the generation of IFN-gamma-producing cytotoxic effectors.     

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T- dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.     

Lymphokine-mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 cells

Murine Th1 and Th2 subsets differ not only in the lymphokines they produce, but also functionally. It is not clear what factors influence the preferential activation of one subset versus the other and what regulatory interactions exist between them. The purpose of this study was to examine the effect of lymphokines produced by clones of Th1 cells (IL-2 and IFN-gamma), Th2 cells (IL-4), and APC (IL-1) on the proliferative response of Th1 and Th2 cells after antigenic stimulation. Activation of both types of clones in the presence of antigen and APC resulted in the acquisition of responsiveness to the proliferative effects of both IL-2 and IL-4, although Th2 cells were more responsive to IL-4 than Th1 cells. Responsiveness of Th1 and Th2 cells to both lymphokines decreased with time after initial antigenic activation; Th1 cells lost their responsiveness to IL-4 more rapidly and to IL-2 more slowly than Th2 cells. IFN-gamma partially inhibited the IL-2 and IL-4-mediated proliferation of Th2, but not Th1 cells. Although the presence of IL-1 was not required for the response of Th1 or Th2 cells to IL-4, its presence resulted in a synergistic effect with IL-2 or IL-4 in Th2 but not in Th1 cells. Both subsets responded to a mixture of IL-2 and IL-4 in synergistic fashion. Delayed addition and wash-out experiments indicated that both IL-2 and IL-4 had to be present simultaneously in order for synergy to occur. These results suggest that Th cell subsets might regulate each other via the lymphokines that they secrete and that the pathways of IL-2 and IL-4 mediated proliferation are interrelated.     

Regulation of T helper type 2 cell differentiation by murine Schnurri-2

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.     

Antigen-pulsed dendritic cells can efficiently induce an antibody response in vivo.

The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low-density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag-pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.     

Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN

The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le- variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.     

Low dose Leishmania major promotes a transient T helper cell type 2 response that is down-regulated by interferon gamma-producing CD8+ T cells

An unresolved issue in the field of T helper (Th) cell development relates to the findings that low doses of antigen promote Th2 cell development in vitro, whereas several classic in vivo studies suggest the opposite. Here we resolve this paradox by studying the early immune response in mice after infection with different doses of Leishmania major. We found that low parasite doses induced a Th2 response in C57BL/6 (B6) mice, whereas high doses induced a Th1 response. However, the Th2 response in low dose-infected mice was transient and the animals healed. The appearance of a Th1 response after low dose infection was dependent upon the concomitant activation of interferon gamma-producing CD8+ T cells. In the absence of CD8+ T cells, the Th2 response was maintained. However, either neutralization of interleukin (IL)-4 or administration of IL-12 promoted a Th1 response after low dose infection of CD8-deficient mice, indicating that the required role for CD8+ T cells was limited to modulation of CD4+ T cell responses. Thus, the discrepant results seen between in vivo and in vitro studies on the effects of antigen dose on Th cell differentiation may depend upon whether CD8+ T cells participate in the immune response.     

Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand

OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin-specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.     

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.     

CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.